(trifluoromethyl)pyrimidine-2-amine compounds

ABSTRACT

The present invention provides a compound of Formula I: 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1  is hydrogen or methyl; and 
             R 2  is: 
           
         
       
    
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt thereof, useful for treating pain, including chronic pain, chronic lower back pain, diabetic peripheral neuropathic pain, and osteoarthritis pain.

The present invention relates to compounds that are potentiators of thehMrgX1 receptor, to pharmaceutical compositions comprising thecompounds, to methods of using the compounds to treat pain, and tointermediates and processes useful in the synthesis of the compounds.

It is estimated that about 20% of adults in the United States alonesuffer from chronic pain. Chronic pain is one of the most common reasonsadults seek medical care and is linked to restrictions in mobility anddaily activities. Unfortunately, chronic pain is often refractory tocurrent therapies and many analgesics are associated with dose-limitingadverse events or serious risk of addiction and abuse which can besubstantial barriers to their use in treating chronic pain. Thus, thereis an unmet need for new chronic pain therapies, particularly treatmentsthat have such adverse effects reduced or effectively eliminated.

U.S. Pat. No. 6,326,368 B1 discloses certain 2-aryloxy- and2-arylthiosubstituted pyrimidines and triazines and derivatives thereofas corticotropin releasing factor (CRF) receptor antagonists useful intreating various disorders, such as depression, anxiety, drug addiction,and inflammatory disorders. U.S. Pat. No. 5,100,459 discloses certainsubstituted sulfonylureas and intermediates thereof. W. Wangdong, et.al., Chem Med Chem, vol 10(1), 57-61 (2015) disclose2-(cyclopropanesulfonamido)-N-(2-ethoxyphenyl)benzamide, ML382, as apotent and selective positive allosteric modulator of MrgX1.

There is a need for alternate treatments of pain including chronic pain.In addition, there is a need for compounds that are potentiators of thehMrgX1 receptor. Accordingly, in one embodiment, the present inventionprovides a compound of Formula I:

-   -   wherein R¹ is hydrogen or methyl; and    -   R² is:

-   -   or a pharmaceutically acceptable salt thereof.        -   In an embodiment, R¹ is hydrogen.        -   In an embodiment, R¹ is methyl.        -   In an embodiment, R² is:

-   -   -   In an embodiment, R² is:

-   -   -   In an embodiment, R² is:

-   -   -   In a particular embodiment, the compound is:

-   -   or a pharmaceutically acceptable salt thereof.        -   In a particular embodiment, the compound is:

-   -   or a pharmaceutically acceptable salt thereof.        -   In a particular embodiment, the compound is:

-   -   or a pharmaceutically acceptable salt thereof.        -   In a particular embodiment, the compound is:

-   -   or a pharmaceutically acceptable salt thereof.        -   In a particular embodiment, the compound is:

-   -   or a pharmaceutically acceptable salt thereof.        -   In a particular embodiment, the compound is:

or a pharmaceutically acceptable salt thereof.

In an embodiment, the present invention also provides a method oftreating pain in a patient in need of such treatment, comprisingadministering to the patient an effective amount of a compound ofFormula I, or a pharmaceutically acceptable salt thereof. In anembodiment, the present invention further provides a method of treatingchronic pain in a patient in need of such treatment, comprisingadministering to the patient an effective amount of a compound ofFormula I, or a pharmaceutically acceptable salt thereof. In anembodiment, the present invention further provides a method of treatingchronic lower back pain in a patient in need of such treatment,comprising administering to the patient an effective amount of acompound of Formula I, or a pharmaceutically acceptable salt thereof. Inan embodiment, the present invention further provides a method oftreating diabetic peripheral neuropathic pain in a patient in need ofsuch treatment, comprising administering to the patient an effectiveamount of a compound of Formula I, or a pharmaceutically acceptable saltthereof. In an embodiment, the present invention further provides amethod of treating osteoarthritis pain in a patient in need of suchtreatment, comprising administering to the patient an effective amountof a compound of Formula I, or a pharmaceutically acceptable saltthereof.

In an embodiment, the present invention further provides a compound ofFormula I, or a pharmaceutically acceptable salt thereof for use intherapy. In an embodiment, the present invention provides a compound ofFormula I, or a pharmaceutically acceptable salt thereof for use intreating pain. In an embodiment, the present invention provides acompound of Formula I, or a pharmaceutically acceptable salt thereof,for use in treating chronic pain.

In an embodiment, the present invention also provides the use of acompound of Formula I, or a pharmaceutically acceptable salt thereof,for the manufacture of a medicament for treating pain. In an embodiment,the present invention provides the use of a compound of Formula I, or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for treating chronic pain.

In an embodiment, the present invention further provides apharmaceutical composition, comprising a compound of Formula I, or apharmaceutically acceptable salt thereof, with one or morepharmaceutically acceptable carriers, diluents, or excipients. In anembodiment, the present invention further provides a process forpreparing a pharmaceutical composition, comprising admixing a compoundof Formula I, or a pharmaceutically acceptable salt thereof, with one ormore pharmaceutically acceptable carriers, diluents, or excipients. Inan embodiment, the present invention also encompasses novelintermediates and processes for the synthesis of compounds of Formula I.

As used herein, the terms “treating”, “treatment”, or “to treat”includes restraining, slowing, stopping, or reversing the progression orseverity of an existing symptom or disorder.

As used herein, the term “patient” refers to a mammal, in particular ahuman.

As used herein, the term “effective amount” refers to the amount or doseof compound of the invention, or a pharmaceutically acceptable saltthereof which, upon single or multiple dose administration to thepatient, provides the desired effect in the patient under diagnosis ortreatment.

An effective amount can be determined by one skilled in the art by theuse of known techniques and by observing results obtained underanalogous circumstances. In determining the effective amount for apatient, a number of factors are considered by the attendingdiagnostician, including, but not limited to: the species of patient;its size, age, and general health; the specific disease or disorderinvolved; the degree of or involvement or the severity of the disease ordisorder; the response of the individual patient; the particularcompound administered; the mode of administration; the bioavailabilitycharacteristics of the preparation administered; the dose regimenselected; the use of concomitant medication; and other relevantcircumstances.

The compounds of the present invention are formulated as pharmaceuticalcompositions administered by any route which makes the compoundbioavailable. Most preferably, such compositions are for oraladministration. Such pharmaceutical compositions and processes forpreparing same are well known in the art (See, e.g., Remington: TheScience and Practice of Pharmacy, L. V. Allen, Editor, 22^(nd) Edition,Pharmaceutical Press, 2012).

Certain intermediates described in the following preparations maycontain one or more nitrogen protecting groups. It is understood thatprotecting groups may be varied as appreciated by one of skill in theart depending on the particular reaction conditions and the particulartransformations to be performed. The protection and deprotectionconditions are well known to the skilled artisan and are described inthe literature (See for example “Greene's Protective Groups in OrganicSynthesis”, Fourth Edition, by Peter G. M. Wuts and Theodora W. Greene,John Wiley and Sons, Inc. 2007).

A pharmaceutically acceptable salt of a compound of the invention can beformed, for example, by reaction of an appropriate free base of acompound of the invention, an appropriate pharmaceutically acceptableacid in a suitable solvent such as diethyl ether under standardconditions well known in the art. Additionally, the formation of suchpharmaceutically acceptable salts can occur simultaneously upondeprotection of a nitrogen protecting group. See, for example, Gould, P.L., “Salt selection for basic drugs,” International Journal ofPharmaceutics, 33: 201-217 (1986); Bastin, R. J., et al. “Salt Selectionand Optimization Procedures for Pharmaceutical New Chemical Entities,”Organic Process Research and Development, 4: 427-435 (2000); and Berge,S. M., et al., “Pharmaceutical Salts,” Journal of PharmaceuticalSciences, 66: 1-19, (1977).

Certain abbreviations are defined as follows: “ACN” refers toacetonitrile; “BAM8-22” refers to bovine adrenal medulla peptide 8-22;“Cat. #” refers to catalog number; “CRC” refers toconcentration-response curve; “DMEM” refers to Dulbecco's modified eaglemedia; “DMSO” refers to dimethyl sulfoxide; “DPBS” refers to Dulbecco'sphosphate-buffered saline; “EC50” refers to the effective concentrationof an agent that gives a half-maximal response between baseline andmaximum after a specified exposure time; “EDTA” refers toethylenediaminetetraacetic acid; “ESMS” refers to Electrospray MassSpectrometry; “FBS” refers to fetal bovine serum; “g” refers to gram orgrams; “h” refers to hour or hours; “HEC” refers tohydroxyethylcellulose; “HEK293” refers to human embryonic kidney 293cell or cells; “HEPES” refers to(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); “hMrgX1” refers tohuman MrgX1 receptor; “HTRF” refers to homogeneous time resolvedfluorescence; “IP 1” refers to inositol monophosphate; “K_(p,uu)” refersto unbound brain-to-plasma partition coefficient; “LC-ESMS” refers torefers to Liquid Chromatography Electrospray Mass Spectrometry; “min”refers to minute or minutes; “mL” refers to milliliter or milliliters;“Me” refers to methyl; “mol” refers to mole or moles; “mmol” refers tomillimole or millimoles; “nm” refers to nanometer or nanometers; “nmol”refers to nanomoles; “m/z” refers to mass-to-charge ration for massspectroscopy; “n,” when in the context of biological data, refers to thenumber of runs or number of times tested; “PBS” refers tophosphate-buffered saline; “rpm” refers to revolutions per minute orminutes; “SD” refers to standard deviation; “SEM” refers to standarderror of the mean; “U/mL” refers to units per milliliter.

General Chemistry

The compounds of the present invention, or salts thereof, may beprepared by a variety of procedures known to one of ordinary skill inthe art, some of which are illustrated in the schemes, preparations, andexamples below. One of ordinary skill in the art recognizes that thespecific synthetic steps for each of the routes described may becombined in different ways, or in conjunction with steps from differentschemes, to prepare compounds of the invention, or salts thereof. Theproducts of each step in the schemes below can be recovered byconventional methods well known in the art, including extraction,evaporation, precipitation, chromatography, filtration, trituration, andcrystallization. In the schemes below, all substituents unless otherwiseindicated, are as previously defined. The reagents and startingmaterials are readily available to one of ordinary skill in the art. Thefollowing schemes, preparations, examples, and assays further illustratethe invention, but should not be construed to limit the scope of theinvention in any way.

Scheme 1 depicts a general preparation of compounds of Formula I (R¹═Hor CH₃; R²=2,6-difluorophenyl, 2,4,6-trifluorophenyl, or4-cyano-2,6-difluorophenyl) via nucleophilic aromatic substitution, asis well known to a person of ordinary skill in the art. Additionally,compounds of Formula I may be prepared via transition-metal (e.g.,copper-, nickel-, or palladium-mediated) cross-coupling or Ullmann-typereactions as is well described in the art.

The 2-amino-6-trifluoromethyl-5-substituted pyrimidine starting materialwith an appropriate leaving group (LG) at the 4-position (e.g., LG=Cl,Br, I, triflate, mesylate, tosylate) may be purchased commercially.Alternatively, a person of ordinary skill in the art will recognize thatthe appropriate starting material may be prepared under a variety oftechniques well documented in the art, such as dehydrative cyclizationof guanidine with an appropriate 5-substituted4,4,4-trifluoro-2-methyl-3-oxo-butanoic acid ester (R¹═H, CH₃) underbasic conditions. Conversion of the subsequent cyclized2-amino-6-trifluoromethyl-4-hydroxy-5-substituted pyrimidine to anappropriate LG at the 4-position is well recognized to a person ofordinary skill in the art.

PREPARATIONS AND EXAMPLES

The following Preparations and Examples further illustrate the inventionand represent typical synthesis of the compound of the invention. Thereagents and starting materials are readily available or may be readilysynthesized by one of ordinary skill in the art. It should be understoodthat the Preparations and Examples are set forth by way of illustrationand not limitation, and that various modifications may be made by one ofordinary skill in the art.

LC-ES/MS is performed on an AGILENT® HP1100 liquid chromatographysystem. Electrospray mass spectrometry measurements are performed on aMass Selective Detector quadrupole mass spectrometer interfaced to theHP1100 HPLC. LC-MS conditions (low pH): column: PHENOMENEX® GEMINI® NXC18 2.1×50 mm 3.0 μm; gradient: 5-100% B in 3 min, then 100% B for 0.75min column temperature: 50° C.+/−10° C.; flow rate: 1.2 mL/min; SolventA: deionized water with 0.1% HCOOH; Solvent B: ACN with 0.1% formicacid; wavelength 214 nm. Alternate LC-MS conditions (high pH): column:XTERRA® MS C18 columns 2.1×50 mm, 3.5 μm; gradient: 5% of solvent A for0.25 min, gradient from 5% to 100% of solvent B in 3 min and 100% ofsolvent B for 0.5 min or 10% to 100% of solvent B in 3 min and at 100%of solvent B for 0.75 min; column temperature: 50° C.+/−10° C.; flowrate: 1.2 mL/min; Solvent A: 10 mM NH₄HCO₃ pH 9; Solvent B: ACN;wavelength: 214 nm.

Preparation 14-(4-Bromo-2,6-difluoro-phenoxy)-6-(trifluoromethyl)pyrimidin-2-amine

Combine 2-amino-4-chloro-6-(trifluoromethyl)pyrimidine (500 mg, 2.4mmol) and 4-bromo-2,6-difluorophenol (616 mg, 2.9 mmol) to a microwavevial and add ACN (10 mL). Add potassium carbonate (665 mg, 4.8 mmol),seal the vial, and heat at 160° C. for 1 h in a microwave reactor. Coolthe reaction mixture, dilute with water, and extract three times withethyl acetate. Combine the organic extracts and dry over sodium sulfate.Filter and evaporate the resulting filtrate under reduced pressure.Purify the resulting residue by flash chromatography over silica gel,using a gradient of 5-100% ethyl acetate in hexanes, to afford the titlecompound (789 mg, 89% yield), after solvent evaporation of the desiredchromatographic fractions. ESMS (m/z, ⁷⁹Br/⁸¹Br): 370/372 [M+H].

Preparation 2 2-Amino-5-methyl-6-(trifluoromethyl)pyrimidin-4-ol

Add a 25% solution of sodium methoxide in methanol (5.5 mL, 24 mmol) toa solution of ethyl 4,4,4-trifluoro-2-methyl-3-oxo-butanoate (4 g, 20mmol) and guanidine (1.2 g, 20 mmol) in methanol (100 mL). Stir for 23 hat room temperature. Evaporate the reaction mixture under reducedpressure. Dissolve the resulting white solid in water (20 mL) andacidify with acetic acid (2 mL). Collect the resulting product by vacuumfiltration, wash twice with water, and dry the resulting filter cakeunder vacuum to obtain the title compound (2.3 g, 60% yield) as a whitesolid. ESMS (m/z): 194 [M+H].

Preparation 3 4-Chloro-5-methyl-6-(trifluoromethyl)pyrimidin-2-amine

In a microwave vial, add phosphoryl chloride (5.5 mL, 59 mmol) to2-amino-5-methyl-6-(trifluoromethyl)pyrimidin-4-ol (2.3 g, 11.9 mmol).Heat the reaction mixture at 110° C. for 30 min in a microwave reactor.Pour the reaction mixture onto ice, basify with 5 M aqueous NaOH (50mL), and extract with ethyl acetate (100 mL). Dry the combined extractsover sodium sulfate, filter, and evaporate the resulting filtrate underreduced pressure. Dissolve the resulting residue in dichloromethane andpurify by flash chromatography over silica gel, eluting with 10-25%ethyl acetate in hexanes, to obtain the title compound (813 mg, 32%yield) as a white solid, after evaporation of the desiredchromatographic fractions. ESMS (m/z, ³⁵Cl/³⁷Cl): 212/214 [M+H].

Preparation 4 4-Chloro-5-iodo-6-(trifluoromethyl)pyrimidin-2-amine

Combine 4-chloro-6-(trifluoromethyl)pyrimidin-2-amine (5 g, 25.2 mmol)in acetic acid (300 mL) with N-iodosuccinimide (32.0 g, 138 mmol) at 0°C. Heat and stir the resulting mixture at 70° C. overnight. Cool thereaction and quench with water (120 mL). Extract with ethyl acetate (100mL×2). Wash the combined organic layers with brine (50 mL×2), dry overNa₂SO₄, filter, and concentrate under reduced pressure. Purify the crudeproduct by flash chromatography to afford 4.8 g (53% yield) of the titleproduct as a yellow solid. ES/MS m/z 324[M+H]⁺

Preparation 54-((2-Amino-5-iodo-6-(trifluoromethyl)pyrimidin-4-yl)oxy)-3,5-difluorobenzonitrile

Combine 4-chloro-5-iodo-6-(trifluoromethyl)pyrimidin-2-amine (2.5 g, 7.0mmol, 90 mass %) and 3,5-difluoro-4-hydroxy-benzonitrile (1.33 g, 8.49mmol) in DMF (35 mL) and add potassium carbonate (2.88 g, 20.8 mmol).Heat the reaction mixture at 90° C. for 3 h. Cool the reaction mixtureand quench with water (100 mL). Extract with ethyl acetate (60 mL×2) andwash the combined organic layers with brine (50 mL×2), dry over Na₂SO₄,filter, and concentrate under reduced pressure. Purify the crude productwith flash chromatography to afford 2.6 g (76% yield) of the titleproduct as a white solid. ES/MS m/z 443[M+H]⁺

EXAMPLE 1 4-(2,6-Difluorophenoxy)-6-(trifluoromethyl)pyrimidin-2-amine

In a microwave vial, add potassium tert-butoxide (270 mg, 2.4 mmol) to asolution of 2-amino-4-chloro-6-(trifluoromethyl)pyrimidine (395 mg, 2.0mmol) and 2,6-difluorophenol (289 mg, 2.2 mmol) in ACN (8.0 mL). Heatthe reaction mixture at 120° C. for 30 min in a microwave reactor.Filter the reaction mixture through diatomaceous earth and evaporate thefiltrate under reduced pressure. Dissolve the resulting residue indichloromethane containing a small amount of methanol and purify themixture by flash chromatography over silica gel, eluting with a gradientof 5-20% ethyl acetate in hexanes, to obtain the title compound (512 mg,88% yield) as a white crystalline solid, after solvent evaporation ofthe desired chromatographic fractions. ESMS (m/z): 292 [M+H].

EXAMPLE 24-(2,4,6-Trifluorophenoxy)-6-(trifluoromethyl)pyrimidin-2-amine

In a 500-mL round-bottom flask, add potassium carbonate (26.9 g, 194.7mmol) to a solution of 2-amino-4-chloro-6-(trifluoromethyl)pyrimidine(19.55 g, 97 mmol) and 2,4,6-trifluorophenol (15.2 g, 97.5 mmol) inN,N-dimethylformamide (200 mL). Heat the reaction mixture at 80° C. for16 h. Quench the reaction mixture with water (500 mL) and extract withethyl acetate (2×500 mL). Wash the combined organic extracts withsaturated aqueous NaCl (2×800 mL), dry over sodium sulfate, filter, andconcentrate the resulting filtrate under reduced pressure. Purify theresulting residue by flash chromatography over silica gel, eluting witha gradient of 0-37% ethyl acetate in petroleum ether, to obtain thetitle compound (15.9 g, 53% yield) as a yellow solid, after evaporationof the desired chromatographic fractions. ESMS (m/z): 310 [M+H].

EXAMPLE 34-[2-Amino-6-(trifluoromethyl)pyrimidin-4-yl]oxy-3,5-difluoro-benzonitrile

Combine4-(4-bromo-2,6-difluoro-phenoxy)-6-(trifluoromethyl)pyrimidin-2-amine(300 mg, 0.8 mmol), zinc cyanide (291 mg, 2.4 mmol),tetrakis(triphenylphosphine)palladium (0) (188 mg, 162 nmol) in amicrowave vial and add N,N-dimethylformamide (6 mL). Seal the vial andheat to 100° C. overnight in a heating block. Cool the reaction, dilutewith water, and extract three times with ethyl acetate. Combine theorganic extracts and dry over sodium sulfate. Filter and evaporate theresulting filtrate under reduced pressure. Purify the resulting residueby flash chromatography over silica gel, using a gradient of 5-100%ethyl acetate in hexanes, to afford the title compound (200 mg, 78%yield), after solvent evaporation of the desired chromatographicfractions. ESMS (m/z): 317 [M+H].

EXAMPLE 45-Methyl-4-(trifluoromethyl)-6-(2,4,6-trifluorophenoxy)pyrimidin-2-amine

In a microwave vial, add potassium tert-butoxide (69 mg, 0.6 mmol) to asolution of 4-chloro-5-methyl-6-(trifluoromethyl)pyrimidin-2-amine (106mg, 0.5 mmol) and 2,4,6-trifluorophenol (84 mg, 0.6 mmol) in ACN (2.0mL). Heat the reaction mixture at 120° C. for 30 min in a microwavereactor. Filter the reaction mixture and evaporate the resultingfiltrate under a stream of air. Dissolve the resulting residue in 1:1dichloromethane/methanol and purify by flash chromatography over silicagel, eluting with 5-10% ethyl acetate/hexanes, to obtain the titlecompound (154 mg, 95% yield) as an off-white solid, after solventevaporation of the desired chromatographic fractions. ESMS (m/z): 324[M+H].

EXAMPLE 54-[2-Amino-5-methyl-6-(trifluoromethyl)pyrimidin-4-yl]oxy-3,5-difluoro-benzonitrile

Combine4-((2-amino-5-iodo-6-(trifluoromethyl)pyrimidin-4-yl)oxy)-3,5-difluorobenzonitrile(1.2 g, 2.4 mmol, 90% purity) and trimethylboroxine (2.5 g, 10 mmol, 50%mass) in 1,4-dioxane (25 mL) and then add cesium carbonate (2.4 g, 7.4mmol) and tetrakis(triphenylphosphine)palladium (0) (580 mg, 0.486855mmol) and heat the reaction to 120° C. for 2 h under nitrogen. Cool thereaction mixture and quench with water (50 mL) and extract with ethylacetate (50 mL×2). Combine the organic layers and wash with brine (30mL×2). Dry the organic layer over sodium sulfate, filter, andconcentrate under reduced pressure. Purify the crude product by flashsilica gel chromatography initially and then further purify by prep-HPLC(Instrument DD, Method Column Xtimate C18 150*40 mm*10 um, Conditionwater (10 mM NH₄HCO₃)-ACN Begin B 50%, End B 80%, with a 10 minutegradient time (min) 10,100% B, Hold Time (min) 2, Flow Rate 60 mL/min).The afforded flows are combined, concentrated to remove most of CH₃CNand then lyophilized to afford 481 mg (60% yield) of the title compoundas a white solid. ES/MS (m/z): 331 (M+H).

EXAMPLE 64-(2,6-difluorophenoxy)-5-methyl-6-(trifluoromethyl)pyrimidin-2-amine

In a microwave vial, add potassium tert-butoxide (69 mg, 0.6 mmol) to asolution of 4-chloro-5-methyl-6-(trifluoromethyl)pyrimidin-2-amine (106mg, 0.5 mmol) and 2,6-difluorophenol (72 mg, 0.6 mmol) in acetonitrile(2.0 mL). Heat the reaction mixture at 120° C. for 30 min in a microwavereactor. Filter the reaction mixture and evaporate the resultingfiltrate under a stream of air. The crude product is purified by reversephase chromatography to obtain the title compound (124 mg, 81% yield) asa white solid. ESMS (m/z): 306 [M+H].

IP1 Cellular Assay for EC₅₀ Determination Against hMrgX1 by HTRF

Cell plating: HEK293 cells stably expressing the recombinant human MrgX1receptor are expanded in culture flasks (Corning, T150), using growthmedia containing DMEM with glutamine (GIBCO™, Cat. #11960-044)supplemented with 10% heat-inactivated FBS (HyClone™, Cat. #CH30073), 1%penicillin/streptomycin (HyClone™, Cat. #SV30010; 10,000 U/mLpenicillin; 10,000 μg/mL streptomycin in 0.85% NaCl), 20 mM HEPES(GIBCO™, Cat. #15530122) and 0.3 mg/mL G418 (GIBCO™, Cat. #11811031).When cell monolayers achieve a level of 80-90% confluence, monolayersare washed once with 10 mL of DPBS (HyClone™, Cat. #14190-144),dissociated using TrypLE™ Express enzyme cell dissociation media(GIBCO™, Cat. #12605-010), and diluted by addition of 10 mL DPBS.Dissociated cells are transferred to a sterile 50 mL conical tube,pelleted by centrifugation at 300×g to remove the growth anddissociation media, and diluted to 1M cells/mL into DMEM for plating.

IP1 Potency and Efficacy Determination: Test compounds are dissolved inDMSO to a concentration of 10 mM and serially diluted in DMSO to obtaina 10-point concentration response stock dilution plate. Growth media isremoved from the cell plate, and the stock 10-point dilution plate isdiluted into media and stamped into the cell plate at a concentration 2×higher than the final test concentration of 30 μM maximum. Theendogenous agonist BAMS-22 (Tocris-BioScience® Cat. #1763) is diluted tothe EC₁₅, determined independently at a minimum of n=3, into the cellplate and incubated at room temperature for 120 min. Subsequently, halfthe volume each of anti-IP1 cryptate and d2-labeled IP1 in lysis buffer,supplied with the IP-One Gq Kit (CisBio Cat. #62IPAPEC) are added to thecell plate to initiate cell lysis, and incubated for 60 min at roomtemperature in the dark. At that point, fluorescence is determined at620 and 665 nm (˜100 μs following laser excitation).

Data Analysis: Fluorescent ratios are determined as the ratio of thefluorescence emission at 620 nm over 665 nm and converted to IP1concentration, using the IP1 standard curve generated in a separateplate, following the manufacturer's instructions. The IP1 concentrationis then plotted as a function of compound concentration. Potentiatorpotency (EC50) is defined as the compound concentration, in the presenceof the EC's of the endogenous agonist BAMS-22, resulting in 50% of theincrease in IP1 concentration achieved by a saturating concentration ofBAMS-22, and is determined by using Genedata software (GeneData AG,Basel Switzerland) fitting the following equation to the 10-point CRC,where y is the IP1 concentration determined for a given compoundconcentration, [L] denotes the concentration of test compound and Max isthe maximum increase achieved by a saturating concentration of BAMS-22:

Y=Max*[L]/(EC ₅₀+[L])

EC50 values are reported as the geometric mean in nM (SEM, n).

TABLE 1 Relative EC₅₀ against hMrgXI IP-1 for the compounds of Examples1-6 Example Relative EC₅₀ (SEM, n) (nM) Max (Mean ± SEM) (nM) 1 72 (30,4)  98 ± 5.3, n = 4 2 61 (26, 4) 109 ± 9.2, n = 4 3 104 (26, 13) 109 ±2.5, n = 13 4 40 (8, 5) 125 ± 7.9, n = 5 5 82, n = l 120 6 38 (3, 3) 103± 1.9, n = 3

Table 1 shows the relative EC50 and the maximum stimulation achieved inthe assay for the compounds of Examples 1 to 6, indicating thesecompounds are potentiators of hMrgX1.

In Vivo Determination of K_(p,uu,brain) in Mice

Unbound brain-to-plasma partition coefficient (K_(p,uu,brain)) is one ofthe key pharmacokinetic parameter for evaluating a compound's ability tocross the blood-brain barrier (BBB). It is typically measured inpre-clinical species using the following methodology. K_(p,uu,brain)values indicate the fraction of free drug in plasma that partitionsacross the BBB.

Subjects: The subjects for these studies are 12 male C57Bl/6 mice(Envigo, Indianapolis, Ind., USA) between 8-10 weeks old at time oftest. Mice are housed in groups of 4 in high density plastic home cages.Food and water is available ad libitum. The rooms are maintained at 73°F. with 30-70% relative humidity and kept on a light/dark cycle of0600-1800 h.Agent: The compound of Example 2 is prepared at 0.3, 1, and 3 mg/ml inthe 1% HEC, 0.25% TWEEN®80, 0.05% DOWSIL™ vehicle in water. Preparedcompound is sonicated in water bath for 30 min until a suspension isformed. Mice are dosed at 10 ml/kg for a respective 3, 10 or 30 mg/kgdose.Dosing and Tissue Collection: For this experiment, four mice per dosinggroup receive oral dosing of either: 3, 10, or 30 mg/kg of the compoundof Example 2. Mice are euthanized at 2 h post-dosing via CO2asphyxiation, plasma samples are collected via cardia puncture, andmouse brains removed, weighed, and frozen on dry ice. Blood samples arestored in EDTA tubes on wet ice and centrifuged at 15 k rpm for 10 min.Plasma is collected, plated in a 96-well plate, and frozen at −80° C.

Pharmacokinetic sampling: Plasma and brain samples obtained are analyzedfor Example 2 using an LC-MS/MS method (Q2 Solutions, Indianapolis,Ind., USA). Plasma samples are extracted using protein precipitation.The lower limit of quantification is 25 ng/mL, and the upper limit ofquantification is 5000 ng/mL. Brain samples are homogenized, and theanalyte is extracted using protein precipitation. The lower limit ofquantification is 4 ng/g and the upper limit of quantification is 200000ng/g.

Determination of plasma and brain protein binding: Mouse plasma andbrain homogenate protein binding is determined in vitro usingequilibrium dialysis, as described elsewhere [Zamek-Gliszczynski et al.,J Pharm Sci, 101:1932-1940, 2012]. The results are reported as fractionunbound in plasma (f_(u,plasma)) and brain (f_(u,brain)) which are thenutilized to calculate K_(p,uu,brain) as described below. Mousef_(u,plasma) and f_(u,brain) of Example 2 are determined to be 0.0421and 0.0181, respectively.

Analysis and Results: K_(p,uu,brain) is calculated for each time pointfrom the expression below where individual components are derived from acombination of in vitro and in vivo measurements carried out asdescribed above:

$K_{p,{uu},{brain}} = {\frac{C_{u,{brain}}}{C_{u,{plasma}}} = {\frac{C_{{total},{brain}}}{C_{{total},{plasma}}} \cdot \frac{f_{u,{brain}}}{f_{u,{plasma}}}}}$

where C_(total,brain), C_(u,brain), C_(total,plasma), and C_(u,plasma)are total and unbound brain and plasma concentrations, and f_(u,brain)and f_(u,plasma) are fractions unbound in brain and plasma,respectively.

TABLE 2 Plasma and brain concentrations of Example 2 post 3, 10, and 30mg/kg oral dose in mouse. Unbound Unbound Total brain plasma Total brainplasma conc. conc. Time Dose conc. conc. (C_(u,brain)) (C_(u,plasma))point Group (C_(total,brain)) (C_(total,plasma)) (nM)* ± (nM){circumflexover ( )} ± (Hours) (mg/kg) (nM) ± SD (nM) ± SD SD SD K_(p,uu,brain) 2 3 972 ± 353 809 ± 140 17.6 ± 6.39 34.1 ± 5.94 0.506 ± 0.107 2 10  3900 ±1800 2770 ± 651  55.4 ± 32.5  117 ± 27.4 0.586 ± 0.116 2 30 13700 ± 42009700 ± 2410  248 ± 75.9 409 ± 101 0.601 ± .0553 *Using mouse f_(u,brain)value of 0.0181 and {circumflex over ( )}mouse f_(u,plasma) value of0.0421, as described above.

The unbound plasma concentration and unbound brain concentration showdose related increases in both plasma and brain indicating the compoundof Example 2 crosses the blood brain barrier and has central penetranceat 2 h post-oral administration in mice. There appears to be doseproportionality in plasma & brain exposure across the dose groups. Meanunbound brain to unbound plasma ratio (Kp,uu-brain) for the compound ofExample 2 ranges from 0.506±0.1 to 0.601±0.0553 (mean±SD, n=4 pergroup); suggesting that an active transport mechanism is not operativein brain tissue in mouse.

We claim:
 1. A compound of the formula:

wherein R¹ is hydrogen or methyl; and R² is:

or a pharmaceutically acceptable salt thereof.
 2. The compound accordingto claim 1 wherein R¹ is hydrogen, or a pharmaceutically acceptable saltthereof.
 3. The compound according to claim 1 wherein R¹ is methyl, or apharmaceutically acceptable salt thereof.
 4. The compound according toclaim 1 wherein R² is:

or a pharmaceutically acceptable salt thereof.
 5. The compound accordingto claim 1 wherein R² is:

or a pharmaceutically acceptable salt thereof.
 6. The compound accordingto claim 1 wherein R² is:

or a pharmaceutically acceptable salt thereof.
 7. The compound accordingto claim 1 wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 8. The compound accordingto claim 7 which is:


9. The compound according to claim 1 wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 10. The compoundaccording to claim 9 which is:


11. The compound according to claim 1 wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 12. The compoundaccording to claim 11 which is:


13. The compound according to claim 1 wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 14. The compoundaccording to claim 13 which is:


15. The compound according to claim 1 wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 16. The compoundaccording to claim 15 which is:


17. The compound according to claim 1 wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 18. The compoundaccording to claim 17 wherein the compound is:


19. A method of treating pain in a patient, comprising administering toa patient in need of such treatment an effective amount of a compoundaccording to claim 1, or a pharmaceutically acceptable salt thereof. 20.A method of treating chronic pain in a patient, comprising administeringto a patient in need of such treatment an effective amount of a compoundaccording to claim 1, or a pharmaceutically acceptable salt thereof. 21.A method of treating chronic lower back pain in a patient, comprisingadministering to a patient in need of such treatment an effective amountof a compound according to claim 1, or a pharmaceutically acceptablesalt thereof.
 22. A method of treating diabetic peripheral neuropathicpain in a patient, comprising administering to a patient in need of suchtreatment an effective amount of a compound according to claim 1, or apharmaceutically acceptable salt thereof.
 23. A method of treatingosteoarthritis pain in a patient, comprising administering to a patientin need of such treatment an effective amount of a compound according toclaim 1, or a pharmaceutically acceptable salt thereof.
 24. Apharmaceutical composition, comprising a compound or a pharmaceuticallyacceptable salt thereof, according to claim 1 with one or morepharmaceutically acceptable carriers, diluents, or excipients.
 25. Aprocess for preparing a pharmaceutical composition, comprising admixinga compound or a pharmaceutically acceptable salt thereof according toclaim 1 with one or more pharmaceutically acceptable carriers, diluents,or excipients.